This invention relates to production of amino acids using auxotrophic mutants of Bacillus methanolicus.
Microorganisms that utilize one-carbon compounds more reduced than carbon dioxide (methylotrophs) are diverse and ubiquitous. Anthony, The Biochemistry of Methylotrophs, page 3 (Academic Press, London 1982); Hanson, Adv. Appl. Microbiol., 26: 3 (1980). Those methylotrophic bacteria reported to utilize methane are all gram-negative and nearly all have an obligate requirement for one-carbon compounds as energy sources. Anthony, supra; Whittenburg et al. J. Gen. Microbiol., 61: 219-226 (1970). Bacteria that grow on methanol and methylamines but not methane include several facultative as well as obligate methylotrophs. Anthony, supra; Hanson, supra. All the obligate methylotrophs unable to utilize methane are gram-negative aerobic bacteria. Anthony, supra.; Whittenburg, supra. Of the facultative methylotrophs isolated that utilize methanol, methylamine or both, only a few were gram positive and were assigned to the genera Bacillus, Corynebacterium, Arthrobacter, or Nocardia. Akiba et al, J. Ferment. Technol., 48: 323-328 (1970); Clement et al. Abstracts of the Fifth International Symposium Microbiol. Growth on C.sub.1 Compounds, p. 69 (Free Univ. Press, Amsterdam 1986); Hazen et al, Arch. Microbiol., 135: 205-210 (1983); Mimura et al., J. Ferment. Technol., 56: 243-252 (1978).
Some species of facultative gram positive methyltrophs that utilize methanol, methylamine or both have now been classified together and named Bacillus methanolicus. Arfman et al., Int. J. System. Bact., 42: 438 (1992). Strains of Bacillus methanolicus have the following identifying characteristics: (1) cells are rod shaped, non-motile, and gram positive; (2) sporulating cells possess oval spores in a subterminal to central position; (3) growth is obligately aerobic at temperatures between 35 to 60.degree. C. with optimum growth at 55.degree. C.; and (4) all strains exhibit growth on methanol. Other characteristics of Bacillus methanolicus are identified in Arfman et al., cited supra.
Production of single cell protein and selected amino acids by microbial fermentation is known, e.g., U.S. Pat. No. 4,652,527 to Stirling. One amino acid which has been produced on an industrial scale is lysine, see Tosaka et al., Trends in Biotechnology, 1: 70-74 (1983), Tosaka and Takinami, Progress in Industrial Microbiology, Ch. 24, pp. 152-172 (Aida et al., 1986). Another example is glutamic acid which has been produced using bacteria of the genera Corynebacterium, Brevibacterium, Microbacterium, and Arothrobacter by fermentation on molasses and starch hydrozylates. Aspartic acid and alanine are produced by enzymatic means from fumaric acid and ammonia. Bacillus species have been used in fermentation processes to produce amino acids, Tosaka et al., supra.; Tosaka and Takinami, supra. However, to date no production of amino acids using an isolated Bacillus species capable of rapid growth on methanol at temperatures above 50.degree. C. has occurred.
The industrial advantages of a thermophilic methanol utilizing fermentation process at elevated temperatures have been described, Snedecor and Cooney, Appl. Microbiol., 27: 112-1117 (1974). For example, use of elevated temperatures can significantly reduce cooling costs. Use of methanol as a carbon and energy source is cost efficient because of its wide availability and low cost. A methanol utilizing, thermophilic mixed culture that included an endospore-forming species was selected by Snedecor and Cooney; however, Snedecor and Cooney, were unable to isolate a pure culture capable of growth on methanol. It is extremely difficult or impossible to isolate appropriate mutants from mixed or impure cultures.
Large scale production of amino acids including glutamic and aspartic acids is desired for many commercial applications. For example, glutamic acid is used in the production of moisturizers in cosmetics, gelatinizing agents in vegetable oils, for oil dispersion, and as a seasoner for foods. The demand for monosodium glutamate exceeds 300,000 tons per year. L-aspartic acid is used as a flavoring agent and to produce the sweetener Aspartame.TM.. The widespread use of this sweetener has rapidly increased demand for L-aspartic acid.
Accordingly, there is a need for a method of producing amino acids using a type I methylotrophic bacterium of the genus Bacillus which exhibits sustained growth on methanol at a temperature of at 50.degree. C. There is also a need for an inexpensive method of producing an amino acid such as glutamic acid on an industrial scale.